The innate, or germline encoded, immune system is an ancient and evolutionary conserved defense system mediating pathogen recognition in humans, animals, and plants. Some innate immune receptors recognize conserved microbial features. Scientific studies have revealed that similar microbial features are recognized across diverse hosts as a result of convergent evolution. For example, in animals, humans and plants bacterial flagellin is recognized by leucine-rich repeat receptors leading to the downstream activation of MAPK cascades and activation of disease resistance. Intracellular immune receptors possessing nucleotide binding, leucine- rich repeat domains (NLRs) act to recognize pathogen effector proteins delivered into plant cells. In humans and animals, NLR receptors can recognize microbial features and, in some cases, effectors. Despite the involvement of analogous immune receptors across diverse organisms, the molecular mechanisms controlling receptor activation and interconnected downstream signaling nodes remain elusive. This is likely due to functional redundancy and lethality underlying key signaling sectors. In this application, we seek to understand the early events underlying NLR receptor activation in plants using the mustard relative, Arabidopsis thaliana, and the bacterial pathogen Pseudomonas syringae as a model. The application will focus on RIN4, a conserved plant protein that can regulate aspects of perception of microbial features, can interact with the NLR receptors RPM1 and RPS2, and is targeted by multiple pathogen effectors. We are able to purify biologically active RPM1 and associated proteins, which will be used to biochemically, investigate NLR receptor activation in vitro. The RPM1 complex will be isolated from Arabidopsis at a resting and activated state and associated proteins will be identified by mass spectrometry. We have optimized conditions for RPM1 complex purification and have several interesting signaling proteins in hand for downstream biochemical, cell biological and genetic validation experiments. The molecular mechanisms controlling effector-induced RIN4 phosphorylation and cleavage for promoting pathogen virulence in susceptible genotypes will be investigated. A greater understanding of how plant immune receptors recognize pathogens will fundamentally advance our understanding of receptor recognition across diverse organisms and is required for the development of novel, environmentally friendly disease control strategies. The specific aims of the project are: 1. Identify changes in complex assembly, enzymatic activity, and known protein associations upon activation of the immune receptor RPM1 in vitro. 2. Identify and characterize NLR protein complexes in a resting and activated state. 3. Investigate the importance of effector-induced RIN4 modifications for interaction with host proteins.